Cd3 binding antibodies

ABSTRACT

The present invention relates to human CD3 antigen-binding polypeptides and their preparation and use in the treatment and/or diagnosis of various diseases, and also relates to bispecific antibody molecules capable of activating immune effector cells and their use in diagnosis and/or treatment of various diseases.

CROSS REFERENCE

This application claims benefit of U.S. Provisional Patent ApplicationNo. 62/394,360, filed Sep. 14, 2016 and U.S. Provisional PatentApplication No. 62/491,908, filed Apr. 28, 2017, which applications areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in XML format and is hereby incorporated by reference inits entirety. Said XML document, created on Nov. 18, 2022, is namedTNO-0010-D2-US2.xml and is 48,000 bytes in size.

BACKGROUND

The body's immune system serves as a defense against infection, injuryand cancer. Two separate but interrelated systems, humoral and cellularimmune systems, work together to protect the body. The humoral system ismediated by soluble factors, named antibodies, which neutralize productsrecognized as being foreign by the body. In contrast, the cellularsystem involves cells, such as T cells and macrophages, which remove andneutralize foreign invaders.

The activation of T cells is critical for the stimulation of immuneresponses. T cells exhibit immunological specificity and direct most ofthe cellular immune responses. Although T cells do not secreteantibodies, they are required for the secretion of antibodies by Blymphocytes. T cell activation requires the participation of a number ofcell surface molecules, such as the T cell receptor complex, and CD4 orCD8 molecules. The antigen-specific T cell receptor (TcR) is composed ofa disulfide-linked heterodimer, membrane glycoprotein with chains, alphaand beta (α and β), or gamma and delta (γ and δ). The TcR isnon-covalently linked with a complex of invariant proteins, designatedCD3.

T cells are known to exert potent antitumor effects in numerousexperimental settings. Antibodies capable of effectively recruiting Tcells against tumor cells have been available as bispecific antibodies,for example directed to tumor-associated antigens (TAAs) and agonisticT-cell membrane proteins, such as the TCR/CD3 complex and CD28. Thesebispecific antibodies are capable of activating T cells, irrespective oftheir TCR specificity, resulting in specific lysis of cells carrying therespective TAAs.

However, while anti-CD3 bispecific antibodies can redirectT-cell-mediated lysis toward malignant cells, clinical trials withCD3-based bsAbs have shown high toxicity in patients. Non-specificT-cell activation from bsAbs can occur in an antigen-independent mannerdue to the Fc/Fc receptor (FcR) interaction, or in an antigen-dependentmanner when antigen is expressed on both normal and tumor cells. Bothmechanisms may have been responsible for the toxicity observed in priorclinical studies. (See for example, Link et al. (1998) Int. J. Cancer77(2):251-6; Durben et al. Molecular Therapy (2015); 23 4, 648-655).Because of the resulting cytokine release syndrome, there have beensignificant blocks to the development of these antibodies fortherapeutic purposes.

The interaction of the T cell receptor (TCR) with its peptide-MHC liganddetermines the activity of a T cell. The binding characteristics of thisinteraction has been studied in great detail and shown to control T cellfunction. The strength and nature of the TCR-peptide/MHC interactiondetermines whether T cells exert effector functions or are inactivatedand deleted. Antibodies against CD3 activate T cells by changing theconformation of the CD3ε chain and depending on the epitope may haveeither agonistic or antagonistic effects on T cells (Yoon et al., 1994Immunity 1:563-569). In light of the significant side-effects of many Tcell agonists it may be preferred to maintain potent anti-tumor effectswhile reducing the release of pro-inflammatory cytokines. However,partial agonistic anti-CD3 antibodies may alter the CD3ε chainsub-optimally resulting in ineffective signaling, and most anti-CD3antibodies are full agonists for both pathways. It is unclear whetherthese effector functions can be separated. Many existing anti-CD3antibodies (for example SP-34, UCHT1, OKT3) have affinities in the rangeof 1-50 nM KD, however this may not be optimal for therapeutic use.

CD3 specific antibodies, and bispecific antibodies derived therefrom areprovided by the invention.

Publications

CD3 antibodies are disclosed, for example, in U.S. Pat. Nos. 5,585,097;5,929,212; 5,968,509; 6,706,265; 6,750,325; 7,381,803; 7,728,114.Bispecific antibodies with CD3 binding specificity are disclosed, forexample, in U.S. Pat. Nos. 7,262,276; 7,635,472; 7,862,813; and8,236,308, each herein specifically incorporated by reference.

SUMMARY

Compositions and methods of use thereof are provided for a family ofclosely related antibodies that bind to and activate signaling throughCD3, e.g. activation of CD3⁺ T cells. The antibody family comprises aset of CDR sequences as defined herein. The family of antibodiesprovides a number of benefits that contribute to utility as clinicallytherapeutic agent(s). The antibodies within the family include memberswith a range of binding affinities, allowing the selection of a specificsequence with a desired affinity. The ability to fine tune affinity isof particular importance to manage the level of CD3 activation in anindividual being treated, and thereby reduce toxicity.

In some embodiments, anti-CD3 antibodies have an affinity (KD) for CD3ranging from around about 10⁻⁶ to around about 10⁻¹¹. Anti-CD3antibodies that have affinities (KD) of 50 nM or greater, 100 nM orgreater, 500 nM or greater, or 1 μM or greater can be desirable to moreclosely mimic the TCR/MHC interaction and minimize toxic cytokinerelease while maintaining effective tumor cell lysis. In someembodiments, anti-CD3 antibodies are characterized or selected forreduced propensity to induce cytokine release, upon binding to acompetent T cell, e.g. for release of IL-2 and IFNγ. Antibodies may beselected for therapeutic use that optimize killing of tumor cells andreduced release of cytokines, e.g. an antibody that, within the familyof antibody sequences described herein, induces a cytokine release thatis less than about half the maximum observed for a family member in acomparative assay, and may be less, e.g. less and about 25% the maximumobserved for a family member in a comparative assay. In someembodiments, bispecific or multispecific antibodies are provided, whichcomprise at least a heavy chain variable region from the antibody familyand may comprise a heavy and light chain variable region providedherein. Bispecific antibodies comprise at least the heavy chain variableregion of an antibody specific for a protein other than CD3, and maycomprise a heavy and light chain variable region. In some suchembodiments, the second antibody specifically binds to a tumorassociated antigen, a targeting antigen, e.g. integrins, etc., apathogen antigen, a checkpoint protein, and the like. Various formats ofbispecific antibodies are within the ambit of the invention, includingwithout limitation single chain polypeptides, two chain polypeptides,three chain polypeptides, four chain polypeptides, and multiplesthereof.

Each of the CD3 specific antibodies comprises a VH domain, comprisingCDR1, CDR2 and CDR3 sequences in a human VH framework. The family 2 CDRsequences may be situated, as an example, in the region of around aminoacid residues 26-33; 51-58; and 97-112 for CDR1, CDR2 and CDR3,respectively, of the provided exemplary variable region sequences setforth in SEQ ID NO: 1-18. It will be understood by one of skill in theart that the CDR sequences may be in different position if a differentframework sequence is selected, although generally the order of thesequences will remain the same.

The CDR sequences for a family 2 antibody may have the followingsequence formulas. An X indicates a variable amino acid, which may bespecific amino acids as indicated below.

CDR1 (SEQ ID NO: 23) G₁ F₂ T₃ F₄ X₅ X6 Y₇ A₈

-   -   where:    -   X₅ may be any amino acid; in some embodiments X₅ is D, A or H;        in some embodiments X₅ is D.    -   X₆ may be any amino acid; in some embodiments X₆ is D or N; in        some embodiments D₆ is D.    -   In some embodiments a CDR1 sequence of a family 2 anti-CD3        antibody comprises the sequence set forth in any of SEQ ID        NO:1-18, residues 26-33.

CDR2 (SEQ ID NO: 24)I_(1′) S_(2′) W_(3′) N_(4′) S_(5′) G_(6′) S_(7′) I_(8′)

-   -   In some embodiments a CDR2 sequence of a family 2 anti-CD3        antibody comprises the sequence set forth in any of SEQ ID NO:        1-18, residues 51-58.

CDR3 (SEQ ID NO: 25)A_(1″) K_(2″) D_(3″) S_(4″) R_(5″) G_(6″) Y_(7″) G_(8″) X_(9″) Y_(10″) X_(11″) X_(12″) G_(13″) G_(12″)A_(15″) Y_(16″)

-   -   where:    -   X_(9″) may be any amino acid, in some embodiments X_(9″) is D or        S; in some embodiments X_(9″) is D;    -   X_(11″) may be any amino acid, in some embodiments X_(11″) is R        or S;    -   X_(12″) may be any amino acid, in some embodiments X_(12″) is L        or R;    -   where:    -   X_(9″) is D;    -   X_(11″) is R or S;    -   X_(12″) is L or R (SEQ ID NO: 48).

In some embodiments a CD3 sequence of a family 2 anti-CD3 antibody hasthe formula A K D S R G Y G D Y X_(11″) X_(12″) G G A Y (SEQ ID NO: 26)where X_(11″) and X_(12″) are as defined above. In some embodiments aCDR3 sequence of a family 2 anti-CD3 antibody comprises the sequence setforth in any of SEQ ID NO: 1-18, residues 97-112. In some embodimentsthe CD3-binding VH domain of a family 2 antibody is paired with a lightchain variable region domain. In some such embodiments the light chainis a fixed light chain.

In some embodiments the light chain comprises a VL domain with CDR1,CDR2 and CDR3 sequences in a human VL framework. The CDR sequences maybe those of SEQ ID NO:19. In some embodiments, the CDR1 sequencecomprises amino acid residues 27-32; 50-52; 89-97 for CDR1, CDR2, CDR3,respectively.

In some embodiments the CDR sequences of an antibody of the inventionare a sequence with at least 85% identity, at least 90% identity, atleast 95% identity, at least 99% identity relative to a CDR sequence orset of CDR sequences in SEQ ID NO: 1-18. In some embodiments a CDRsequence of the invention comprises one, two, three or more amino acidsubstitutions relative to a CDR sequence or set of CDR sequences in anyone of SEQ ID NO: 1-18. In some embodiments said amino acidsubstitution(s) are one or more of position 5 or 10 of CDR1, position 2,6 or 7 of CDR2, position 1, 8, 9 or 10 of CDR3, relative to the family 2formulas provided above.

In some embodiments, a bispecific antibody of the invention comprises aCD3-binding variable region described herein, paired with a light chain.In some embodiments the light chain comprises the variable regionsequence set forth in SEQ ID NO:19, or a variable region comprising theset of CDR sequences in SEQ ID NO:19 and framework sequences. Various Fcsequences find use, including without limitation human IgG1, IgG2a,IgG2b, IgG3, IgG4, etc. In some embodiments, the second arm of thebispecific antibody comprises a variable region that specifically bindsto a tumor-associated antigen. In some embodiments, the second arm ofthe bispecific antibody comprises a variable region that specificallybinds to BCMA. In some embodiments the anti-BCMA arm is a single chainvariable region, for example as shown in FIG. 2B. In some embodimentsthe anti-BCMA arm comprises the variable region sequence set forth inSEQ ID NO:20; or the tandem variable region sequence set forth in SEQ IDNO:21. The Fc sequence of the anti-BCMA arm may be, without limitation,human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. The CDR sequences may bethose contained in SEQ ID NO:20. In some embodiments, the CDR sequencecomprises amino acid residues 26-33; 51-58; 97-108 for CDR1, CDR2, CDR3,respectively.

In other embodiments, pharmaceutical compositions are provided,comprising at least a

CD3-binding VH domain of the invention, e.g. a monospecific, bispecific,etc. antibody or antibody-like protein comprising at least a CD3-bindingVH domain of the invention; and a pharmaceutically acceptable excipient.The composition may be lyophilized, suspended in solution, etc. and maybe provided in a unit dose formulation.

In some embodiments, a method is provided for treatment of cancer, themethod comprising administering to an individual in need thereof aneffective dose of a mono-specific, bi-specific, etc. antibody of theinvention. Where the antibody is bispecific, a second antigen-bindingsite may specifically bind a tumor antigen, a checkpoint protein, etc.In various embodiments, the cancer is selected from the group consistingof ovarian cancer, breast cancer, gastrointestinal, brain cancer, headand neck cancer, prostate cancer, colon cancer, lung cancer, leukemia,lymphoma, sarcoma, carcinoma, neural cell tumors, squamous cellcarcinomas, germ cell tumors, metastases, undifferentiated tumors,seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors, andneoplasias caused by infectious agents.

In some embodiments, a method is provided for treatment of infectiousdisease, the method comprising administering to an individual in needthereof an effective dose of a mono-specific, bi-specific, etc. antibodyof the invention. Where the antibody is bispecific, a secondantigen-binding site may specifically bind a pathogen antigen, e.g.bacteria, viruses or parasites.

In other embodiments, a method is provided for the production of abispecific antibody of the present invention comprising expressing theantibody sequences, e.g. one or more light chain encoding sequences, oneor more heavy chain encoding sequences, in a single host cell. Invarious embodiments, the host cell may be a prokaryotic or an eukaryoticcell, such as a mammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1C. FIG. 1A shows an alignment of CDR1, 2 and 3 regions ofmembers of antibody family 2 of SEQ ID NO:1-18, which specifically bindto human CD3, corresponding to residues 26-33; 51-58; and 97-112. FIG.1B shows the CDR1, 2 and 3 regions of the fixed light chain (SEQ IDNO:19); and an exemplary anti-BCMA sequence (SEQ ID NO:20 and SEQ IDNO:21). FIG. 1C provides the CDR sequences of a reference anti-CD3antibody (SEQ ID NO:22), ID 304704. FIG. 1A discloses the CDR1 sequencesas SEQ ID NOS 29, 29, 29, 29, 29, 30, 31, 32, 33, 33, 29, 29, 34, 29,33, 29, 29 and 29, the CDR2 sequence “ISWNSGSI” as SEQ ID NO: 24, andthe CDR3 sequences as SEQ ID NOS 41, 41, 42, 43, 43, 42, 42, 42, 42, 42,42, 44, 43, 42, 43, 41, 41 and 41, all respectively, in order ofappearance. FIG. 1B discloses the CDR1 sequences as SEQ ID NOS 35, 36and 36, the CDR2 sequences as SEQ ID NOS 38, 39 and 39, and the CDR3sequences as SEQ ID NOS 45, 46 and 46, all respectively, in order ofappearance. FIG. 1C discloses SEQ ID NOS 37, 40 and 47, respectively, inorder of appearance.

FIG. 2A-2E. Schematic models of bispecific human antibodies. FIG. 2Aanti-CD3:anti-tumor-antigen bispecific antibody with common light chain(3 total unique chains). FIG. 2B anti-CD3:anti-tumor-antigen bispecificantibody with 2 unique light chains (4 total unique chains). FIG. 2Canti-CD3:anti-tumor-antigen bispecific antibody with heavy-chain onlytumor antigen binding domain chain (3 unique chains). FIG. 2Danti-CD3:anti-tumor-antigen bispecific antibody with scFv tumor antigenbinding domain (3 total unique chains). FIG. 2Eanti-CD3:anti-tumor-antigen bispecific antibody with scFv anti-CD3binding domain (3 total unique chains)

FIG. 3 . Anti-CD3 family 2 data table summarizes the behavior ofanti-CD3 antibodies in monospecific and bispecific format. Column 1shows the sequence ID for the anti-CD3 VH sequence. Column 2 shows theMFI value for Jurkat cell binding of the parental monospecific anti-CD3.Column 3 shows the MFI value for cyno T-cell binding of the parentalmonospecific anti-CD3. Column 4 shows the name of the aCD3:aBCMAbispecific antibody. Column 5 shows the picograms of IL-2 released bypan T-cells stimulated by the bispecific antibody binding the BCMAprotein coated on plastic at the dose indicated. Column 6 shows thepicograms of IL-6 released by pan T-cells stimulated by the bispecificantibody binding the BCMA protein coated on plastic at the doseindicated. Column 7 shows the picograms of IL-10 released by pan T-cellsstimulated by the bispecific antibody binding the BCMA protein coated onplastic at the dose indicated. Column 8 shows the picograms of IFN-γreleased by pan T-cells stimulated by the bispecific antibody bindingthe BCMA protein coated on plastic at the dose indicated. Column 9 showsthe picograms of TNFα released by pan T-cells stimulated by thebispecific antibody binding the BCMA protein coated on plastic at thedose indicated. Column 10 shows the EC₅₀ of bispecific antibody-mediatedU266 tumor cell lysis in presence of human pan T-cells. Column 11 showsthe percent lysis of U266 tumor cells in the presence of bispecificantibody and human pan T-cells at a dose of 333 ng/mL of bispecificantibody. Column 12 shows the protein binding affinity of the anti-CD3arm of the bispecific antibody measured by Octet. Column 13 shows theMFI value for Jurkat cell binding of the bispecific antibody.

FIG. 4 . Bispecific antibody-mediated tumor cell lysis. SevenαCD3_fam2:aBCMA bispecific antibodies, each with a unique anti-CD3 armand a common anti-BCMA arm, were tested for the ability to kill U266BCMA+ tumor cells through redirection of activated primary T cells. Inthis experiment U266 cells that express BCMA were mixed with activatedpan T-cells in a 10:1 E:T ratio along with the addition of bispecificantibody. The x-axis shows the concentration of antibody used and they-axis shows the % lysis of tumor cells 6 hours after addition ofantibody.

FIG. 5 . Bispecific U266 killing activity correlated with IL-2 release.A comparison of bispecific antibody-mediated tumor cell lysis activitywith IL-2 cytokine release is shown in the scatter plot. The correlationbetween IL-2 production and U266 tumor cell lysis is R²=0.37.

FIG. 6 . Bispecific U266 killing activity correlated with IFN-γ release.A comparison of bispecific antibody-mediated tumor cell lysis activitywith IFN-γ cytokine release is shown in the scatter plot. Thecorrelation between IFN-g production and U266 tumor cell lysis isR²=0.53.

FIG. 7 . Bispecific U266 killing activity correlated with anti-CD3binding affinity. A comparison of bispecific antibody-mediated U266tumor cell lysis activity with anti-CD3 binding affinity is shown in thescatter plot. The correlation between U266 killing EC50 and proteinbinding affinity is R²=0.93.

FIG. 8A-8D. Bispecific antibody-mediated tumor cell lysis.αCD3_F1F:aBCMA bispecific antibodies were assayed for the ability tokill three different BCMA+ tumor cells and one BCMA-negative cell linethrough redirection of activated primary T cells. In this experiment,tumor cells were mixed with activated pan T-cells in a 10:1 E:T ratioalong with the addition of bispecific antibody. FIG. 8A shows killing ofRPMI-8226 cells, FIG. 8B shows killing of NCI-H929 cells, FIG. 8C showskilling of U-266 cells, and FIG. 8D shows killing of K562 cells, anegative control. The x-axis shows the concentration of antibody usedand the y-axis shows the % lysis of tumor cells 6 hours after additionof antibody.

FIG. 9A-9D. Bispecific antibody-mediated IL-2 release. The level of IL-2cytokine release was measured after resting human T cells were culturedwith various tumor cell lines and increasing doses of αCD3_F1F:aBCMAbispecific antibody. FIG. 9A shows IL-2 release stimulated by RPMI-8226cells, FIG. 9B shows IL-2 release stimulated by NCI-H929 cells, FIG. 9Cshows IL-2 release stimulated by U-266 cells, and FIG. 9D shows IL-2release stimulated by K562 cells, a negative control.

FIG. 10A-10D. Bispecific antibody-mediated IFN-γ release. The level ofIFN-γ cytokine release was measured after resting human T cells werecultured with various tumor cell lines and increasing doses ofαCD3_F1F:aBCMA bispecific antibody. FIG. 10A shows IFN-γ releasestimulated by RPMI-8226 cells, FIG. 10B shows IFN-γ release stimulatedby NCI-H929 cells, FIG. 10C shows IFN-γ release stimulated by U-266cells, and FIG. 10D shows IFN-γ release stimulated by K562 cells, anegative control.

DETAILED DESCRIPTION

To facilitate an understanding of the invention, a number of terms aredefined below.

Before the present active agents and methods are described, it is to beunderstood that this invention is not limited to the particularmethodology, products, apparatus and factors described, as such methods,apparatus and formulations may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adrug candidate” refers to one or mixtures of such candidates, andreference to “the method” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

Generally, conventional methods of protein synthesis, recombinant cellculture and protein isolation, and recombinant DNA techniques within theskill of the art are employed in the present invention. Such techniquesare explained fully in the literature, see, e.g., Maniatis, Fritsch &Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook,Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001);Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: PortableProtocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory;(1988).

Definitions

By “comprising” it is meant that the recited elements are required inthe composition/method/kit, but other elements may be included to formthe composition/method/kit etc. within the scope of the claim.

By “consisting essentially of”, it is meant a limitation of the scope ofcomposition or method described to the specified materials or steps thatdo not materially affect the basic and novel characteristic(s) of thesubject invention.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease. Thetherapeutic agent may be administered before, during or after the onsetof disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy may be administered during the symptomatic stage ofthe disease, and in some cases after the symptomatic stage of thedisease.

A “therapeutically effective amount” is intended for an amount of activeagent which is necessary to impart therapeutic benefit to a subject. Forexample, a “therapeutically effective amount” is an amount whichinduces, ameliorates or otherwise causes an improvement in thepathological symptoms, disease progression or physiological conditionsassociated with a disease or which improves resistance to a disorder.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having cancer, individuals with autoimmune diseases, withpathogen infections, and the like. Subjects may be human, but alsoinclude other mammals, particularly those mammals useful as laboratorymodels for human disease, e.g. mouse, rat, etc.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. Cells of interestfor detection, analysis, or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. Cancers of virtually every tissue are known. Thephrase “cancer burden” refers to the quantum of cancer cells or cancervolume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g. clone of a cancercell. Many types of cancers are known to those of skill in the art,including solid tumors such as carcinomas, sarcomas, glioblastomas,melanomas, lymphomas, myelomas, etc., and circulating cancers such asleukemias, including specifically B cell leukemias, T cell leukemias,etc. Examples of cancer include but are not limited to, ovarian cancer,breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, headand neck cancer, and brain cancer.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors, such as natural killer cells, neutrophils, andmacrophages, recognize bound antibody on a target cell and cause lysisof the target cell. ADCC activity may be assessed using methods, such asthose described in U.S. Pat. No. 5,821,337. ADCP refers to antibodydependent cell-mediated phagocytosis.

“Effector cells” are leukocytes which express one or more constantregion receptors and perform effector functions.

A “cytokine” is a protein released by one cell to act on another cell asan intercellular mediator. Cytokines of interest include, withoutlimitation, cytokines released from activated T cells, for example IL-2,IFNγ, etc.

“Non-immunogenic” refers to a material that does not initiate, provokeor enhance an immune response where the immune response includes theadaptive and/or innate immune responses.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

“Homology” between two sequences is determined by sequence identity. Iftwo sequences, which are to be compared with each other, differ inlength, sequence identity preferably relates to the percentage of thenucleotide residues of the shorter sequence which are identical with thenucleotide residues of the longer sequence. Sequence identity can bedetermined conventionally with the use of computer programs such as theBestf it program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive Madison, Wis. 53711). Bestfit utilizes the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2(1981), 482-489, in order to find the segment having the highestsequence identity between two sequences. When using Bestfit or anothersequence alignment program to determine whether a particular sequencehas for instance 95% identity with a reference sequence of the presentinvention, the parameters are preferably so adjusted that the percentageof identity is calculated over the entire length of the referencesequence and that homology gaps of up to 5% of the total number of thenucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at theirpreset (“default”) values. The deviations appearing in the comparisonbetween a given sequence and the above-described sequences of theinvention may be caused for instance by addition, deletion,substitution, insertion or recombination. Such a sequence comparison canpreferably also be carried out with the program “fasta20u66” (version2.0u66, September 1998 by William R. Pearson and the University ofVirginia; see also W. R. Pearson (1990), Methods in Enzymology 183,63-98, appended examples and http://workbench.sdsc.edu/). For thispurpose, the “default” parameter settings may be used.

“Variant” refers to polypeptides having amino acid sequences that differto some extent from a native sequence polypeptide. Ordinarily, aminoacid sequence variants will possess at least about 80% sequenceidentity, more preferably, at least about 90% homologous by sequence.The amino acid sequence variants may possess substitutions, deletions,and/or insertions at certain positions within the reference amino acidsequence.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they are operablylinked. Such vectors are referred to herein as “recombinant expressionvectors” (or simply, “recombinant vectors”). In general, expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids. In the present specification, “plasmid” and “vector” may beused interchangeably as the plasmid is the most commonly used form ofvector.

The term “host cell” (or “recombinant host cell”), as used herein, isintended to refer to a cell that has been genetically altered, or iscapable of being genetically altered by introduction of an exogenouspolynucleotide, such as a recombinant plasmid or vector. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody or other binding molecule) and its binding partner(e.g., an antigen or receptor). The affinity of a molecule X for itspartner Y can generally be represented by the dissociation constant(Kd). Affinity can be measured by common methods known in the art,including those described herein. Low-affinity antibodies bind antigen(or receptor) weakly and tend to dissociate readily, whereashigh-affinity antibodies bind antigen (or receptor) more tightly andremain bound longer.

Unless specifically indicated to the contrary, the term “conjugate” asdescribed and claimed herein is defined as a heterogeneous moleculeformed by the covalent attachment of one or more antibody fragment(s) toone or more polymer molecule(s), wherein the heterogeneous molecule iswater soluble, i.e. soluble in physiological fluids such as blood, andwherein the heterogeneous molecule is free of any structured aggregate.A conjugate of interest is PEG. In the context of the foregoingdefinition, the term “structured aggregate” refers to (1) any aggregateof molecules in aqueous solution having a spheroid or spheroid shellstructure, such that the heterogeneous molecule is not in a micelle orother emulsion structure, and is not anchored to a lipid bilayer,vesicle or liposome; and (2) any aggregate of molecules in solid orinsolubilized form, such as a chromatography bead matrix, that does notrelease the heterogeneous molecule into solution upon contact with anaqueous phase. Accordingly, the term “conjugate” as defined hereinencompasses the aforementioned heterogeneous molecule in a precipitate,sediment, bioerodible matrix or other solid capable of releasing theheterogeneous molecule into aqueous solution upon hydration of thesolid.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

Antibodies, also referred to as immunoglobulins, conventionally compriseat least one heavy chain and one light, where the amino terminal domainof the heavy and light chains is variable in sequence, hence is commonlyreferred to as a variable region domain, or a variable heavy (VH) orvariable light (VH) domain. The two domains conventionally associate toform a specific binding region, although as well be discussed here,specific binding can also be obtained with heavy chain only variablesequences, and a variety of non-natural configurations of antibodies areknown and used in the art.

A “functional” or “biologically active” antibody or antigen-bindingmolecule (including heavy chain only antibodies and bispecificthree-chain antibody-like molecules (TCAs) herein) is one capable ofexerting one or more of its natural activities in structural,regulatory, biochemical or biophysical events. For example, a functionalantibody or other binding molecule, e.g. TCA, may have the ability tospecifically bind an antigen and the binding may in turn elicit or altera cellular or molecular event such as signaling transduction orenzymatic activity. A functional antibody or other binding molecule,e.g. TCA, may also block ligand activation of a receptor or act as anagonist or antagonist. The capability of an antibody or other bindingmolecule, e.g. TCA, to exert one or more of its natural activitiesdepends on several factors, including proper folding and assembly of thepolypeptide chains.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,monomers, dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), heavy chain only antibodies, three chain antibodies, singlechain Fv, nanobodies, etc., and also include antibody fragments, so longas they exhibit the desired biological activity (Miller et al (2003)Jour. of Immunology 170:4854-4861). Antibodies may be murine, human,humanized, chimeric, or derived from other species.

The term antibody may reference a full-length heavy chain, a full lengthlight chain, an intact immunoglobulin molecule; or an immunologicallyactive portion of any of these polypeptides, i.e., a polypeptide thatcomprises an antigen binding site that immunospecifically binds anantigen of a target of interest or part thereof, such targets includingbut not limited to, cancer cell or cells that produce autoimmuneantibodies associated with an autoimmune disease. The immunoglobulindisclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule, including engineered subclasses with altered Fcportions that provide for reduced or enhanced effector cell activity.The immunoglobulins can be derived from any species. In one aspect, theimmunoglobulin is of largely human origin.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a beta-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the beta-sheet structure. The hypervariable regions in each chain areheld together in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region may comprise amino acid residues from a“complementarity determining region” or “CDR”, and/or those residuesfrom a “hypervariable loop”. “Framework Region” or “FR” residues arethose variable domain residues other than the hypervariable regionresidues as herein defined.

Variable regions of interest include at least one CDR sequence from thefamily 2 variable regions provided herein, usually at least 2 CDRsequences, and more usually 3 CDR sequences. Exemplary CDR designationsare shown herein, however one of skill in the art will understand that anumber of definitions of the CDRs are commonly in use, including theKabat definition (see “Zhao et al. A germline knowledge basedcomputational approach for determining antibody complementaritydetermining regions.” Mol Immunol. 2010;47:694-700), which is based onsequence variability and is the most commonly used. The Chothiadefinition is based on the location of the structural loop regions(Chothia et al. “Conformations of immunoglobulin hypervariable regions.”Nature. 1989; 342:877-883). Alternative CDR definitions of interestinclude, without limitation, those disclosed by Honegger, “Yet anothernumbering scheme for immunoglobulin variable domains: an automaticmodeling and analysis tool.” J Mol Biol. 2001;309:657-670; Ofran et al.“Automated identification of complementarity determining regions (CDRs)reveals peculiar characteristics of CDRs and B cell epitopes.” JImmunol. 2008;181:6230-6235; Almagro “Identification of differences inthe specificity-determining residues of antibodies that recognizeantigens of different size: implications for the rational design ofantibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al.“Identification of specificity-determining residues in antibodies.”Faseb J. 1995;9:133-139., each of which is herein specificallyincorporated by reference.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod.

The antibodies herein specifically include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984)Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc) and human constant region sequences.

An “intact antibody chain” as used herein is one comprising a fulllength variable region and a full length constant region (Fc). An intact“conventional” antibody comprises an intact light chain and an intactheavy chain, as well as a light chain constant domain (CL) and heavychain constant domains, CH1, hinge, CH2 and CH3 for secreted IgG. Otherisotypes, such as IgM or IgA may have different CH domains. The constantdomains may be native sequence constant domains (e.g., human nativesequence constant domains) or amino acid sequence variants thereof. Theintact antibody may have one or more “effector functions” which refer tothose biological activities attributable to the Fc constant region (anative sequence Fc region or amino acid sequence variant Fc region) ofan antibody. Examples of antibody effector functions include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; anddown regulation of cell surface receptors. Constant region variantsinclude those that alter the effector profile, binding to Fc receptors,and the like.

Depending on the amino acid sequence of the Fc (constant domain) oftheir heavy chains, antibodies and various antigen-binding proteins canbe provided as different classes. There are five major classes of heavychain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA, and IgA2. The Fc constant domains that correspond to thedifferent classes of antibodies may be referenced as α, δ, ε, γ, and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Ig forms include hinge-modifications or hingeless forms (Roux et al(1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem.267:7246-7256; US 2005/0048572; US 2004/0229310). The light chains ofantibodies from any vertebrate species can be assigned to one of twotypes, called κ and λ, based on the amino acid sequences of theirconstant domains.

A “functional Fc region” possesses an “effector function” of anative-sequence Fc region. Exemplary effector functions include C1qbinding; CDC; Fc-receptor binding; ADCC; ADCP; down-regulation ofcell-surface receptors (e.g., B-cell receptor), etc. Such effectorfunctions generally require the Fc region to be interact with areceptor, e.g. the FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA;FcγRIIIB receptors, and the law affinity FcRn receptor; and can beassessed using various assays as disclosed, for example, in definitionsherein. A “dead” Fc is one that has been mutagenized to retain activitywith respect to, for example, prolonging serum half-life, but which doesnot activate a high affinity Fc receptor.

A “native-sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature.Native-sequence human Fc regions include, for example, a native-sequencehuman IgG1 Fc region (non-A and A allotypes); native-sequence human IgG2Fc region; native-sequence human IgG3 Fc region; and native-sequencehuman IgG4 Fc region, as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence that differs fromthat of a native-sequence Fc region by virtue of at least one amino acidmodification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native-sequence Fc region or to the Fc regionof a parent polypeptide, e.g., from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native-sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native-sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

Variant Fc sequences may include three amino acid substitutions in theCH2 region to reduce FcγRI binding at EU index positions 234, 235, and237 (see Duncan et al., (1988) Nature 332:563). Two amino acidsubstitutions in the complement C1q binding site at EU index positions330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med.178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)).Substitution into human IgG1 of IgG2 residues at positions 233-236 andIgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC(see, for example, Armour K L. et al., 1999 Eur J Immunol.29(8):2613-24; and Shields R L. et al., 2001. J Biol Chem.276(9):6591-604). Other Fc variants are possible, including withoutlimitation one in which a region capable of forming a disulfide bond isdeleted, or in which certain amino acid residues are eliminated at theN-terminal end of a native Fc form or a methionine residue is addedthereto. Thus, in one embodiment of the invention, one or more Fcportions of the scFc molecule can comprise one or more mutations in thehinge region to eliminate disulfide bonding. In yet another embodiment,the hinge region of an Fc can be removed entirely. In still anotherembodiment, the molecule can comprise an Fc variant.

Further, an Fc variant can be constructed to remove or substantiallyreduce effector functions by substituting, deleting or adding amino acidresidues to effect complement binding or Fc receptor binding. Forexample, and not limitation, a deletion may occur in acomplement-binding site, such as a C1q-binding site. Techniques ofpreparing such sequence derivatives of the immunoglobulin Fc fragmentare disclosed in International Patent Publication Nos. WO 97/34631 andWO 96/32478. In addition, the Fc domain may be modified byphosphorylation, sulfation, acylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like.

The Fc may be in the form of having native sugar chains, increased sugarchains compared to a native form or decreased sugar chains compared tothe native form, or may be in an aglycosylated or deglycosylated form.The increase, decrease, removal or other modification of the sugarchains may be achieved by methods common in the art, such as a chemicalmethod, an enzymatic method or by expressing it in a geneticallyengineered production cell line. Such cell lines can includemicroorganisms, e.g. Pichia Pastoris, and mammalians cell line, e.g. CHOcells, that naturally express glycosylating enzymes. Further,microorganisms or cells can be engineered to express glycosylatingenzymes, or can be rendered unable to express glycosylation enzymes (Seee.g., Hamilton, et al., Science, 313:1441 (2006); Kanda, et al, J.Biotechnology, 130:300 (2007); Kitagawa, et al., J. Biol. Chem., 269(27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264 (23): 13848(1989); Imai-Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO07/055916). As one example of a cell engineered to have alteredsialylation activity, the alpha-2,6-sialyltransferase 1 gene has beenengineered into Chinese Hamster Ovary cells and into sf9 cells.Antibodies expressed by these engineered cells are thus sialylated bythe exogenous gene product. A further method for obtaining Fc moleculeshaving a modified amount of sugar residues compared to a plurality ofnative molecules includes separating said plurality of molecules intoglycosylated and non-glycosylated fractions, for example, using lectinaffinity chromatography (See e.g., WO 07/117505). The presence ofparticular glycosylation moieties has been shown to alter the functionof Immunoglobulins. For example, the removal of sugar chains from an Fcmolecule results in a sharp decrease in binding affinity to the C1q partof the first complement component C1 and a decrease or loss inantibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC), thereby not inducingunnecessary immune responses in vivo. Additional important modificationsinclude sialylation and fucosylation: the presence of sialic acid in IgGhas been correlated with anti-inflammatory activity (See e.g., Kaneko,et al, Science 313:760 (2006)), whereas removal of fucose from the IgGleads to enhanced ADCC activity (See e.g., Shoj-Hosaka, et al, J.Biochem., 140:777 (2006)).

In alternative embodiments, antibodies of the invention may have an Fcsequence with enhanced effector functions, e.g. by increasing theirbinding capacities to FcyRIIIA and increasing ADCC activity. Forexample, fucose attached to the N-linked glycan at Asn-297 of Fcsterically hinders the interaction of Fc with FcγRIIIA, and removal offucose by glyco-engineering can increase the binding to FcγRIIIA, whichtranslates into >50-fold higher ADCC activity compared with wild typeIgG1 controls. Protein engineering, through amino acid mutations in theFc portion of IgG1, has generated multiple variants that increase theaffinity of Fc binding to FcγRIIIA. Notably, the triple alanine mutantS298A/E333A/K334A displays 2-fold increase binding to FcγRIIIA and ADCCfunction. S239D/I332E (2×) and S239D/I332E/A330L (3×) variants have asignificant increase in binding affinity to FcγRIIIA and augmentation ofADCC capacity in vitro and in vivo. Other Fc variants identified byyeast display also showed the improved binding to FcγRIIIA and enhancedtumor cell killing in mouse xenograft models. See, for example Liu etal. (2014) JBC 289(6):3571-90, herein specifically incorporated byreference.

The term “Fc-region-comprising antibody” refers to an antibody thatcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the antibody or by recombinant engineering thenucleic acid encoding the antibody. Accordingly, an antibody having anFc region according to this invention can comprise an antibody with orwithout K447.

“Fv” is the minimum antibody fragment, which contains a completeantigen-recognition and antigen-binding site. The CD3 binding antibodiesof the invention comprise a dimer of one heavy chain and one light chainvariable domain in tight, non-covalent association; however additionalantibodies, e.g. for use in a multi-specific configuration, may comprisea VH in the absence of a VL sequence. Even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although theaffinity may be lower than that of two domain binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Humanized” forms of non-human (e.g., rodent) antibodies, includingsingle chain antibodies, are chimeric antibodies (including single chainantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. See, for example, Jones et al, (1986) Nature321:522-525; Chothia et al (1989) Nature 342:877; Riechmann et al (1992)J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol.224:487-499; Presta et al (1993) J. Immunol. 151, 2623-2632; Werther etal (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001)Thromb. Haemost. 85:379-389. For further details, see U.S. Pat. Nos.5,225,539; 6,548,640; 6,982,321; 5,585,089; 5,693,761; 6,407,213; Joneset al (1986) Nature, 321:522-525; and Riechmann et al (1988) Nature332:323-329.

The term “single chain antibody” as used herein means a singlepolypeptide chain containing one or more antigen binding domains thatbind an epitope of an antigen, where such domains are derived from orhave sequence identity with the variable region of an antibody heavy orlight chain. Parts of such variable region may be encoded by V_(H) orV_(L) gene segments, D and J_(H) gene segments, or J_(L) gene segments.The variable region may be encoded by rearranged V_(H)DJ_(H),V_(L)DJ_(H), V_(H)J_(L), or V_(L)J_(L) gene segments. V-, D- and J-genesegments may be derived from humans and various animals including birds,fish, sharks, mammals, rodents, non-human primates, camels, lamas,rabbits and the like.

The CD3-binding antibodies of the invention find particular utility inmulti-specific configurations, which include without limitationbispecific antibodies, trifunctional antibodies, etc. A large variety ofmethods and protein configurations are known and use in bispecificmonoclonal antibodies (BsMAB), tri-specific antibodies, etc.

First-generation BsMAbs consisted of two heavy and two light chains, oneeach from two different antibodies. The two Fab regions are directedagainst two antigens. The Fc region is made up from the two heavy chainsand forms the third binding site with the Fc receptor on immune cells(see for example Lindhofer et al., The Journal of Immunology, Vol 155, p219-225, 1995). The antibodies may be from the same or differentspecies. For example, cell lines expressing rat and mouse antibodiessecrete functional bispecific Ab because of preferentialspecies-restricted heavy and light chain pairing. In other embodimentsthe Fc regions are designed to only fit together in specific ways.

Other types of bispecific antibodies include chemically linked Fabs,consisting only of the Fab regions. Two chemically linked Fab or Fab2fragments form an artificial antibody that binds to two differentantigens, making it a type of bispecific antibody. Antigen-bindingfragments (Fab or Fab2) of two different monoclonal antibodies areproduced and linked by chemical means like a thioether (see Glennie, M Jet al., Journal of immunology 139, p 2367-75, 1987; Peter Borchmann etal., Blood, Vol. 100, No. 9, p 3101-3107, 2002).

Various other methods for the production of multivalent artificialantibodies have been developed by recombinantly fusing variable domainsof two antibodies. A single-chain variable fragment (scFv) is a fusionprotein of the variable regions of the heavy (VH) and light chains (VL)of immunoglobulins, connected with a short linker peptide of ten toabout 25 amino acids. The linker is usually rich in glycine forflexibility, as well as serine or threonine for solubility, and caneither connect the N-terminus of the VH with the C-terminus of the VL,or vice versa. Bispecific single-chain variable fragments (di-scFvs,bi-scFvs) can be engineered by linking two scFvs with differentspecificities. A single peptide chain with two VH and two VL regions isproduced, yielding bivalent scFvs.

Bispecific tandem scFvs are also known as bi-specific T-cell engagers(BiTEs). Bispecific scFvs can be created with linker peptides that aretoo short for the two variable regions to fold together (about fiveamino acids), forcing scFvs to dimerize. This type is known as diabodies(Adams et al., British journal of cancer 77, p 1405-12, 1998). TheDual-Affinity Re-Targeting (DART) platform technology (Macrogenics,Rockville, Md.). This fusion protein technology uses two single-chainvariable fragments (scFvs) of different antibodies on a single peptidechain of about 55 kilodaltons. SCORPION Therapeutics (EmergentBiosolutions, Inc., Seattle, Wash.) combines two antigen-binding domainsin a single chain protein. One binding domain is on the C-terminus and asecond binding domain on the N-terminus of an effector domain, based onimmunoglobulin Fc regions.

Tetravalent and bispecific antibody-like proteins also include DVD-Igswhich are engineered from two monoclonal antibodies (Wu, C. et al.,Nature Biotechnology, 25, p 1290-1297, 2007). To construct the DVD-Igmolecule, the V domains of the two mAbs are fused in tandem by a shortlinker (TVAAP (SEQ ID NO: 27)) with the variable domain of the firstantibody light (VL) chain at the N terminus, followed by the otherantibodies VL and Ck to form the DVD-Ig protein light chain. Similarly,the variable regions of the heavy (VH) chain of the two mAbs are fusedin tandem by a short linker (ASTKGP (SEQ ID NO: 28)) with the firstantibody at the N terminus, followed by the other antibody and the heavychain constant domains to form the DVD-Ig protein heavy chain (VH1/VL1).All light chain and heavy chain constant domains are preserved in theDVD-Ig design, as they are critical for the formation of adisulfide-linked full IgG-like molecule. Cotransfection of mammaliancells with expression vectors encoding the DVD-Ig light chain and heavychain leads to the secretion of a single species of an IgG-like moleculewith molecular weight of approximately 200 kDa. This molecule has nowfour binding sites, 2 from each mAb.

The term “bispecific three-chain antibody like molecule” or “TCA” isused herein to refer to antibody-like molecules comprising, consistingessentially of, or consisting of three polypeptide subunits, two ofwhich comprise, consist essentially of, or consist of one heavy and onelight chain of a monoclonal antibody, or functional antigen-bindingfragments of such antibody chains, comprising an antigen-binding regionand at least one CH domain. This heavy chain/light chain pair hasbinding specificity for a first antigen. The third polypeptide subunitcomprises, consists essentially of, or consists of a heavy chain onlyantibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4domains, in the absence of a CH1 domain, and an antigen binding domainthat binds an epitope of a second antigen or a different epitope of thefirst antigen, where such binding domain is derived from or has sequenceidentity with the variable region of an antibody heavy or light chain.Parts of such variable region may be encoded by V_(H) and/or V_(L) genesegments, D and J_(H) gene segments, or J_(L) gene segments. Thevariable region may be encoded by rearranged V_(H)DJ_(H), V_(L)DJ_(H),V_(H)J_(L), or V_(L)J_(L) gene segments.

A TCA protein makes use of a heavy chain only antibody” or “heavy chainantibody” or “heavy chain polypeptide” as used herein means a singlechain antibody comprising heavy chain constant regions CH2 and/or CH3and/or CH4 but no CH1 domain. In one embodiment, the heavy chainantibody is composed of an antigen-binding domain, at least part of ahinge region and CH2 and CH3 domains. In another embodiment, the heavychain antibody is composed of an antigen-binding domain, at least partof a hinge region and a CH2 domain. In a further embodiment, the heavychain antibody is composed of an antigen-binding domain, at least partof a hinge region and a CH3 domain. Heavy chain antibodies in which theCH2 and/or CH3 domain is truncated are also included herein. In afurther embodiment the heavy chain is composed of an antigen bindingdomain, and at least one CH (CH1, CH2, CH3, or CH4) domain but no hingeregion. The heavy chain only antibody can be in the form of a dimer, inwhich two heavy chains are disulfide bonded other otherwise, covalentlyor non-covalently attached with each other. The heavy chain antibody maybelong to the IgG subclass, but antibodies belonging to othersubclasses, such as IgM, IgA, IgD and IgE subclass, are also includedherein. In a particular embodiment, the heavy chain antibody is of theIgG1, IgG2, IgG3, or IgG4 subtype, in particular IgG1 subtype.

Heavy chain antibodies constitute about one fourth of the IgG antibodiesproduced by the camelids, e.g. camels and llamas (Hamers-Casterman C.,et al. Nature. 363, 446-448 (1993)). These antibodies are formed by twoheavy chains but are devoid of light chains. As a consequence, thevariable antigen binding part is referred to as the VHH domain and itrepresents the smallest naturally occurring, intact, antigen-bindingsite, being only around 120 amino acids in length (Desmyter, A., et al.J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with ahigh specificity and affinity can be generated against a variety ofantigens through immunization (van der Linden, R. H., et al. Biochim.Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readilycloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol.78, 11-21 (2000)). Their levels of expression, solubility and stabilityare significantly higher than those of classical F(ab) or Fv fragments(Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks havealso been shown to have a single VH-like domain in their antibodiestermed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003);Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley etal., Molecular Immunology 40, 25-33 (2003)).

An antibody or antigen-binding molecule, including the heavy chain onlyantibodies and bispecific three-chain antibody-like molecules (TCAs)herein, “which binds” an antigen of interest, is one that binds theantigen with sufficient affinity such that the antibody or bindingmolecule is useful as a diagnostic and/or therapeutic agent in targetingthe antigen, and does not significantly cross-react with other proteins.In such embodiments, the extent of binding of the antibody or otherbinding molecule to a non-targeted antigen will be no more than 10% asdetermined by fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA).

Proteins

The present invention provides a family of closely related antibodiesthat bind to and activate signaling through CD3, e.g. activation of CD3⁺T cells. The antibodies within the family comprise a set of CDRsequences as defined herein, and are exemplified by the provided VHsequences of SEQ ID NO:1-18. The family of antibodies provides a numberof benefits that contribute to utility as clinically therapeuticagent(s). The antibodies within the family include members with a rangeof binding affinities, allowing the selection of a specific sequencewith a desired affinity. The ability to fine tune affinity is ofparticular importance to manage the level of CD3 activation in anindividual being treated, and thereby reduce toxicity. For example, iflow abundant tumor antigens (less than 10,000 molecules per cell) aretargeted, it is anticipated that high affinity CD3 binders (<30 nM) arepreferred. If highly abundant tumor antigens (more than 50,000 moleculesper cell) are targeted, CD3 binders with low affinities (>50 nM) arepreferred. Separately evaluated from affinity can be the propensity ofthe antibody to induce release of cytokines when bound to a T cell, e.g.release of IL-2, IFNγ, etc., where reduced cytokine release may bedesirable.

A suitable antibody may be selected from those provided herein fordevelopment and use, including without limitation use as a bispecificantibody. Determination of affinity for a candidate protein can beperformed using methods known in the art, e.g. Biacore measurements,etc. Members of the antibody family may have an affinity for CD3 with aKd of from about 10⁻⁶ to around about 10⁻¹¹, including withoutlimitation: from about 10⁻⁶ to around about 10⁻¹⁰; from about 10⁻⁶ toaround about 10⁻⁹; from about 10⁻⁶ to around about 10⁻⁸; from about 10⁻⁸to around about 10⁻¹¹; from about 10⁻⁸ to around about 10⁻¹⁰; from about10⁻⁸ to around about 10⁻⁹; from about 10⁻⁹ to around about 10⁻¹¹; fromabout 10⁻⁹ to around about 10⁻¹⁰; or any value within these ranges. Theaffinity selection may be confirmed with a biological assessment foractivation of T cells in, for example, and in vitro or pre-clinicalmodel, and assessment of potential toxicity. Determination of cytokinerelease can be evaluated using any convenient method, including withoutlimitation the assays described in the examples.

Engagement of the T cell receptor (TCR), either by binding MH-peptidecomplexes or anti-TCR/CD3 antibodies, initiates T cell activation.Examples of anti-TCR/CD3 antibodies that activate T cells are OKT3 andUCHT1. These anti-CD3 antibodies cross-compete for binding to CD3 on Tcells and are routinely used in T cell activation assays. Anti-CD3antibodies of this invention cross-compete with OKT3 for binding tohuman CD3. Depending on the binding affinity for CD3 and epitope on CD3,anti-CD3 antibodies activated T cells with different functionaloutcomes. In vitro incubation of human T cells with low affinityanti-CD3 antibodies resulted in incomplete activation of T cells, lowIL-2 and IL-10 production. In contrast, high-affinity CD3 bindersactivated T cells to produce significantly more IL-2 and othercytokines. The low-affinity anti-CD3 antibodies are considered partialagonists that selectively induce some effector functions, potent tumorkilling and CD69 upregulation, while failing to induce others, such asIL-2 and IL-10 production. The high-affinity binders of this inventionare full-agonists activating many immune effector functions of T cells.The strength of the interaction with CD3 and the epitope recognizedresulted in qualitatively different activation of T cells. Maximalcytokine production of T cells activated by low-affinity anti-CD3antibodies was lower than maximal activation by high-affinity anti-CD3antibodies. In some embodiments, an antibody of the invention results ina lower release of one or both of IL-2 and IL-10 when combined with Tcells in an activation assay when compared to a reference anti-CD3antibody in the same assay, where the reference antibody can be ID304703 (SEQ ID NO:22) or an antibody of equivalent affinity. The maximalrelease of IL-2 and/or IL-10 can be less than about 75% of the releaseby the reference antibody, less than about 50% of the release by thereference antibody, less than about 25% of the release by the referenceantibody, and may be less than about 10% of the release by a referenceantibody.

In some embodiments of the invention, bispecific or multispecificantibodies are provided, which may have any of the configurationsdiscussed herein, including without limitation a three chain bispecific.Bispecific antibodies comprise at least the heavy chain variable regionof an antibody specific for a protein other than CD3, and may comprise aheavy and light chain variable region. In some such embodiments, thesecond antibody specificity binds to a tumor associated antigen, atargeting antigen, e.g. integrins, etc., a pathogen antigen, acheckpoint protein, and the like. Various formats of bispecificantibodies are within the ambit of the invention, including withoutlimitation single chain polypeptides, two chain polypeptides, threechain polypeptides, four chain polypeptides, and multiples thereof.

The family of CD3 specific antibodies comprise a VH domain, comprisingCDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequencesmay be situated, as an example, in the region of around amino acidresidues 26-33; 51-58; and 97-112 for CDR1, CDR2 and CDR3, respectively,of the provided exemplary variable region sequences set forth in SEQ IDNO:1-18. It will be understood by one of skill in the art that the CDRsequences may be in different position if a different framework sequenceis selected, although generally the order of the sequences will remainthe same.

The CDR sequences for a family 2 antibody may have the followingsequence formulas. An X indicates a variable amino acid, which may bespecific amino acids as indicated below.

CDR1 (SEQ ID NO: 23) G₁ F₂ T₃ F₄ X₅ X6 Y₇ A₈

-   -   where:    -   X₅ may be any amino acid; in some embodiments X₅ is D, A or H;        in some embodiments X₅ iS D.    -   X₆ may be any amino acid; in some embodiments X₆ is D or N; in        some embodiments D₆ is D.    -   In some embodiments a CDR1 sequence of a family 2 anti-CD3        antibody comprises the sequence set forth in any of SEQ ID        NO:1-18, residues 26-33.

CDR2 (SEQ ID NO: 24)I_(1′) S_(2′) W_(3′) N_(4′) S_(5′) G_(6′) S_(7′) I_(8′)

-   -   In some embodiments a CDR2 sequence of a family 2 anti-CD3        antibody comprises the sequence set forth in any of SEQ ID        NO:1-18, residues 51-58.

CDR3 (SEQ ID NO: 25)A_(1″) K_(2″) D_(3″) S_(4″) R_(5″) G_(6″) Y_(7″) G_(8″) X_(9″) Y_(10″) X_(11″) X_(12″) G_(13″) G_(12″)A_(15″) Y_(16″)

-   -   where:    -   X_(9″) may be any amino acid, in some embodiments X_(9″) is D or        S; in some embodiments X_(9″) is D;    -   X_(11″) may be any amino acid, in some embodiments X_(11″) is R        or S;    -   X_(12″) may be any amino acid, in some embodiments X_(12″) is L        or R;    -   where:    -   X_(9″) is D;    -   X_(11″) is R or S;    -   X_(12″) is L or R (SEQ ID NO: 48).        -   In some embodiments a CD3 sequence of a family 2 anti-CD3            antibody has the formula A K D S R G Y G D Y X_(11″) X_(12″)            G G A Y (SEQ ID NO: 26) where X_(11″) and X_(12″) are as            defined above. In some embodiments a CDR3 sequence of a            family 2 anti-CD3 antibody comprises the sequence set forth            in any of SEQ ID NO:1-18, residues 97-112.

In some embodiments the CD3-binding VH domain is paired with a lightchain variable region domain. In some such embodiments the light chainis a fixed light chain. In some embodiments the light chain comprises aVL domain with CDR1, CDR2 and CDR3 sequences in a human VL framework.The CDR sequences may be those of SEQ ID NO:19. In some embodiments, theCDR1 sequence comprises amino acid residues 27-32; 50-52; 89-97 forCDR1, CDR2, CDR3, respectively.

In some embodiments the aCDR sequences of a family 2 antibody have asequence with at least 85% identity, at least 90% identity, at least 95%identity, at least 99% identity relative to a CDR sequence or set of CDRsequences in any one of SEQ ID NO:1-18. In some embodiments a CDRsequence of the invention comprises one, two, three or more amino acidsubstitutions relative to a CDR sequence or set of CDR sequences in anyone of SEQ ID NO:1-18. In some embodiments said amino acidsubstitution(s) are one or more of position 5 or 10 of CDR1, position 2,6 or 7 of CDR2, position 1, 8, 9 or 10 of CDR3, relative to the formulasprovided above.

Where a protein of the invention is a bispecific antibody, one bindingmoiety, i.e. VH/VL combination or VH only, is specific for human CD3while the other arm may be specific for target cells, including cancercells, such as cells of ovarian, breast, gastrointestinal, brain, headand neck, prostate, colon, and lung cancers, and the like, as well ashematologic tumors such as B-cell tumors, including leukemias,lymphomas, sarcomas, carcinomas, neural cell tumors, squamous cellcarcinomas, germ cell tumors, metastases, undifferentiated tumors,seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors,neoplasias caused by infectious agents, and other malignancies, cellsinfected with a pathogen, autoreactive cells causing inflammation and/orautoimmunity. The non-CD3 moiety can also be specific for an immuneregulatory protein, as will be described herein.

Tumor-associated antigens (TAAs) are relatively restricted to tumorcells, whereas tumor-specific antigens (TSAs) are unique to tumor cells.TSAs and TAAs typically are portions of intracellular moleculesexpressed on the cell surface as part of the major histocompatibilitycomplex.

Tissue specific differentiation antigens are molecules present on tumorcells and their normal cell counterparts. Tumor-associated antigensknown to be recognized by therapeutic mAbs fall into several differentcategories. Hematopoietic differentiation antigens are glycoproteinsthat are usually associated with cluster of differentiation (CD)groupings and include CD20, CD30, CD33 and CD52. Cell surfacedifferentiation antigens are a diverse group of glycoproteins andcarbohydrates that are found on the surface of both normal and tumorcells. Antigens that are involved in growth and differentiationsignaling are often growth factors and growth factor receptors. Growthfactors that are targets for antibodies in cancer patients include CEA,epidermal growth factor receptor (EGFR; also known as ERBB1)′ ERBB2(also known as HER2), ERBB3, MET (also known as HGFR), insulin-likegrowth factor 1 receptor (IGF1R), ephrin receptor A3 (EPHA3), tumornecrosis factor (TNF)-related apoptosis-inducing ligand receptor 1(TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B)and receptor activator of nuclear factor-κB ligand (RANKL; also known asTNFSF11). Antigens involved in angiogenesis are usually proteins orgrowth factors that support the formation of new microvasculature,including vascular endothelial growth factor (VEGF), VEGF receptor(VEGFR), integrin αVβ3 and integrin α5β1. Tumor stroma and theextracellular matrix are indispensable support structures for a tumor.Stromal and extracellular matrix antigens that are therapeutic targetsinclude fibroblast activation protein (FAP) and tenascin.

Examples of therapeutic antibodies useful in bispecific configurationsinclude, without limitation, rituximab; Ibritumomab; tiuxetan;tositumomab; Brentuximab; vedotin; Gemtuzumab; ozogamicin; Alemtuzumab;IGN101; adecatumumab; Labetuzumab; huA33; Pemtumomab; oregovomab; CC49(minretumomab); cG250; J591; MOv18; MORAb-003 (farletuzumab); 3F8,ch14.18; KW-2871; hu3S193; IgN311; Bevacizumab; IM-2C6; CDP791;Etaracizumab; Volociximab; Cetuximab, panitumumab, nimotuzumab; 806;Trastuzumab; pertuzumab; MM-121; AMG 102, METMAB; SCH 900105; AVE1642,IMC-A12, MK-0646, R1507; CP 751871; KB004; IIIA4; Mapatumumab(HGS-ETR1); HGS-ETR2; CS-1008; Denosumab; Sibrotuzumab; F19; and 81C6.

The immune-checkpoint receptors that have been most actively studied inthe context of clinical cancer immunotherapy, cytotoxicT-lymphocyte-associated antigen 4 (CTLA4; also known as CD152) andprogrammed cell death protein 1 (PD1; also known as CD279) - are bothinhibitory receptors. The clinical activity of antibodies that blockeither of these receptors implies that antitumor immunity can beenhanced at multiple levels and that combinatorial strategies can beintelligently designed, guided by mechanistic considerations andpreclinical models.

The two ligands for PD1 are PD1 ligand 1 (PDL1; also known as B7-H1 andCD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed oncancer cells and through binding to its receptor PD1 on T cells itinhibits T cell activation/function.

Lymphocyte activation gene 3 (LAGS; also known as CD223), 2B4 (alsoknown as CD244), B and T lymphocyte attenuator (BTLA; also known asCD272), T cell membrane protein 3 (TIM3; also known as HAVcr2),adenosine A2a receptor (A2aR) and the family of killer inhibitoryreceptors have each been associated with the inhibition of lymphocyteactivity and in some cases the induction of lymphocyte anergy. Antibodytargeting of these receptors can be used in the methods of theinvention.

Agents that agonize an immune costimulatory molecule are also useful inthe methods of the invention. Such agents include agonists or CD40 andOX40. CD40 is a costimulatory protein found on antigen presenting cells(APCs) and is required for their activation. These APCs includephagocytes (macrophages and dendritic cells) and B cells. CD40 is partof the TNF receptor family. The primary activating signaling moleculesfor CD40 are IFNγ and CD40 ligand (CD40L). Stimulation through CD40activates macrophages.

Anti CCR4 (CD194) antibodies of interest include humanized monoclonalantibodies directed against C-C chemokine receptor 4 (CCR4) withpotential anti-inflammatory and antineoplastic activities. CCR2 isexpressed on inflammatory macrophages that can be found in variousinflammatory conditions, e.g. rheumatoid arthritis; and have also beenidentified as expressed on tumor promoting macrophages. CCR2 is alsoexpressed on regulatory T cells, and the CCR2 ligand, CCL2, mediatesrecruitment of regulatory T cells into tumors. Regulatory T cellssuppress a response for anti-tumor T cells and thus their inhibition ordepletion is desired.

Producing Proteins of the Invention

Although antibodies can be prepared by chemical synthesis, they aretypically produced by methods of recombinant DNA technology, such asco-expression of all the chains making up the protein in a singlerecombinant host cell, or co-expression of a heavy chain polypeptide andan antibody, e.g. a human antibody. In addition, the antibody heavy andlight chains can also be expressed using a single polycistronicexpression vector. Purification of individual polypeptides is achievedusing standard protein purification technologies such as affinity(protein A) chromatography, size exclusion chromatography and/orhydrophobic interaction chromatography. Bispecifics are sufficientlydifferent in size and hydrophobicity that purification can be performedusing standard procedures.

The amount of antibody and heavy chain polypeptide produced in a singlehost cell can be minimized through engineering of constant regions ofthe antibody and the heavy chain such that homodimerization is favoredover heterodimerization, e.g. by introducing self-complementaryinteractions (see e.g. WO 98/50431 for possibilities, such as“protuberance-into-cavity” strategies (see WO 96/27011)). It istherefore another aspect of the present invention to provide a methodfor producing a bispecific in a recombinant host, the method includingthe step of: expressing in a recombinant host cell a nucleic acidsequences encoding at least two heavy chain polypeptides, wherein saidheavy chain polypeptides differ in their constant regions sufficientlyto reduce or prevent homodimer formation but increase bispecificformation.

Where the protein comprises three chains, e.g. FlicAbs, they may beproduced by co-expression of the three chains (2 heavy chains and onelight chain) making up the molecule in a single recombinant host cell.

For recombinant production of the proteins herein, one or more nucleicacids encoding all chains, e.g. 2, 3 4, etc. are isolated and insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. Many vectors are available. The vector componentsgenerally include, but are not limited to, one or more of the following:a signal sequence, an origin of replication, one or more marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

In a preferred embodiment, the host cell according to the method of theinvention is capable of high-level expression of human immunoglobulin,i.e. at least 1 pg/cell/day, preferably at least 10 pg/cell/day and evenmore preferably at least 20 pg/cell/day or more without the need foramplification of the nucleic acid molecules encoding the single chainsin said host cell.

Pharmaceutical Composition

It is another aspect of the present invention to provide pharmaceuticalcompositions comprising one or more proteins of the present invention inadmixture with a suitable pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers as used herein are exemplified, butnot limited to, adjuvants, solid carriers, water, buffers, or othercarriers used in the art to hold therapeutic components, or combinationsthereof.

Therapeutic formulations of the proteins used in accordance with thepresent invention are prepared for storage by mixing proteins having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (see, e.g. Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as inthe form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Anti-CD3 antibody formulations are disclosed, for example, in U.S.Patent Publication No. 20070065437, the entire disclosure is expresslyincorporated by reference herein. Similar formulations can be used forthe proteins of the present invention. The main components of suchformulations are a pH buffering agent effective in the range of 3.0 to6.2, a salt, a surfactant, and an effective amount of a bispecific withanti-CD3 specificity.

Methods of Use

Methods are provided for treating or reducing disease, including withoutlimitation infection, autoimmune disease, primary or metastatic cancer,etc. in a regimen comprising contacting the targeted cells with anantigen-binding composition of the invention, particularly where theantigen-binding composition is a multi-specific antibody suitable forthe condition being treated, e.g. where one binding moiety specificallybinds to a tumor associated antigen for treatment of the relevant cancercells; a binding moiety specific for a pathogen of interest fortreatment of the relevant infection, and the like. Such methods includeadministering to a subject in need of treatment a therapeuticallyeffective amount or an effective dose of the agents of the invention,including without limitation combinations of the reagent with achemotherapeutic drug, radiation therapy, or surgery.

Effective doses of the compositions of the present invention for thetreatment of disease vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g. companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

Dosage levels can be readily determined by the ordinarily skilledclinician, and can be modified as required, e.g., as required to modifya subject's response to therapy. The amount of active ingredient thatcan be combined with the carrier materials to produce a single dosageform varies depending upon the host treated and the particular mode ofadministration. Dosage unit forms generally contain between from about 1mg to about 500 mg of an active ingredient.

In some embodiments, the therapeutic dosage the agent may range fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once every two weeks or once a month oronce every 3 to 6 months. Therapeutic entities of the present inventionare usually administered on multiple occasions. Intervals between singledosages can be weekly, monthly or yearly. Intervals can also beirregular as indicated by measuring blood levels of the therapeuticentity in the patient. Alternatively, therapeutic entities of thepresent invention can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the polypeptidein the patient.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor growth, tumor metastasis or tumorinvasion of cancers including carcinomas, hematologic cancers such asleukemias and lymphomas, melanomas, sarcomas, gliomas, etc. Forprophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk ofdisease in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease.

Compositions for the treatment of disease can be administered byparenteral, topical, intravenous, intratumoral, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal orintramuscular means. A typical route of administration is intravenous orintratumoral, although other routes can be equally effective.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges. It isrecognized that compositions of the invention when administered orally,should be protected from digestion. This is typically accomplishedeither by complexing the molecules with a composition to render themresistant to acidic and enzymatic hydrolysis, or by packaging themolecules in an appropriately resistant carrier, such as a liposome or aprotection barrier. Means of protecting agents from digestion are wellknown in the art.

The compositions for administration will commonly comprise an antibodyor other ablative agent dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of active agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs (e.g., Remington's Pharmaceutical Science (15th ed.,1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics(Hardman et al., eds., 1996)).

Also within the scope of the invention are kits comprising the activeagents and formulations thereof, of the invention and instructions foruse. The kit can further contain a least one additional reagent, e.g. achemotherapeutic drug, etc. Kits typically include a label indicatingthe intended use of the contents of the kit. The term label includes anywriting, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit.

The compositions can be administered for therapeutic treatment.Compositions are administered to a patient in an amount sufficient tosubstantially ablate targeted cells, as described above. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.”, which may provide for an improvement in overall survival rates.Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. The particular dose required for a treatmentwill depend upon the medical condition and history of the mammal, aswell as other factors such as age, weight, gender, administration route,efficiency, etc.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXAMPLES Example 1 Genetically Engineered Rats Expressing HeavyChain-Only Antibodies

A human IgH locus was constructed and assembled in several parts, whichinvolved the modification and joining of rat C region genes, which werethen joined downstream of human V_(H)6-D-J_(H) region. Two BACs withseparate clusters of human VH genes were then co-injected with a BACencoding the assembled (human V_(H)6-D-J_(H)-rat C) fragment.

Transgenic rats carrying artificial heavy chain immunoglobulin loci inunrearranged configuration were generated. The included constant regiongenes encode IgM, IgD, IgG2b, IgE, IgA and 3′ enhancer. RT-PCR and serumanalysis (ELISA) of transgenic rats revealed productive rearrangement oftransgenic immunoglobulin loci and expression of heavy chain onlyantibodies of various isotypes in serum. Transgenic rats were cross-bredwith rats with mutated endogenous heavy chain and light chain locipreviously described in US patent publication 2009/0098134 A1. Analysisof such animals demonstrated inactivation of rat immunoglobulin heavyand light chain expression and high level expression of heavy chainantibodies with variable regions encoded by human V, D, and J genes.Immunization of transgenic rats resulted in production of high titerserum responses of antigen-specific heavy chain antibodies. Thesetransgenic rats expressing heavy chain antibodies with a human VDJregion were called UniRats.

Example 2 Genetically Engineered Rats Expressing Fixed Light ChainAntibodies

Transgenic human antibody repertoires were generated from H-chains withdiverse (V_(H)-D-J_(H))_(n) rearrangement in combination with a uniqueL-chain. For this a rearranged L-chain, human Vk-Jk1-Ck, was integratedin the rat germline by DNA microinjection and the obtained transgenicanimals were bred with a previously described rat strain that expressesa human H-chain repertoire naturally (Osborn et al., 2013). This new ratstrain was named OmniFlic.

Immunizations of OmniFlic rats, using many different antigens, producedhigh levels of antigen-specific IgG similar to other transgenic ratscarrying the same IgH locus. Repertoire analysis by RT-PCR identifiedhighly variable VH-gene rearrangements at high transcript and proteinlevels. In addition, only one L-chain product, also expressed at highlevel, was identified.

Antigen-specific binders from OmniFlic were obtained by NGS andselection from cDNA libraries (yeast, E.coli, phage), which uponsequencing identified diverse H-chain transcripts. For the expression inmammalian cells hypermutated H-chain constructs were transfected incombination with the original transgenic Igk sequence. In thisrearranged Vk-Jk1-Ck no mutational changes were allowed and always thesame L-chain was expressed with various H-chain products to generatemonoclonal human IgG.

Example 3 Generation of Antigen-Specific Antibodies in Transgenic Rats

For the generation of antigen-specific heavy chain antibodies in rats,genetically engineered rats expressing were immunized in two ways.

Immunization with recombinant extracellular domains of PD-L1 and BCMA.Recombinant extracellular domains of PD-L1 and BCMA were purchased fromR&D Systems and were diluted with sterile saline and combined withadjuvant. Immunogens were either combined with Complete Freund'sAdjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) or Titermax andRibi adjuvants. The first immunization (priming) with immunogen in CFAor Titermax was administered in the left and right legs. After the firstimmunization with immunogens in CFA two more immunizations in IFA(boosters) or 4 more immunizations in Ribi and one more in Titermax wereadministered in each leg. This sequence of immunizations leads to thedevelopment of B cells producing high affinity antibodies. The immunogenconcentrations were 10 microgram per leg. Serum was collected from ratsat the final bleed to determine serum titers.

For the generation of anti-human CD3δε antibodies genetically engineeredrats were immunized using DNA-based immunization protocols.

OmniFlic rats were immunized with human and cynomolgus CD3-epsilon/deltaconstructs at Aldevron, Inc. (Fargo, N. Dak.) using the GENOVAC AntibodyTechnology. Draining lymph nodes were harvested after the final boostand RNA isolated. Following cDNA synthesis, the IgH heavy chain antibodyrepertoire was characterized by Next Generation Sequencing and ourproprietary in-house software. Candidate antigen-specific VH sequencesshowing evidence of antigen-specific positive selection were selected.Several hundred VH sequences encoding FlicAbs were selected for geneassembly and cloned into an expression vector. Subsequently, fully humanFlicAb IgG1 antibodies were expressed in HEK cells for analysis by Flowand ELISA. Human FlicAbs were tested for binding to primary human Tcells and Jurkat cells by flow. In addition, human FlicAbs were testedusing recombinant CD3δε proteins in ELISA. All FlicAbs with positivebinding for human T cells are listed in FIGS. 1 and 2 . Selectedsequences were further characterized in T cell activation assays.

Example 4 Characterization of Antibodies

The data table of FIG. 3 summarizes the behavior of anti-CD3 antibodiesof family 2 in monospecific and bispecific format. Column 1 shows thesequence ID for the anti-CD3 VH sequence. Column 2 shows the MFI valuefor Jurkat cell binding of the parental monospecific anti-CD3. Column 3shows the MFI value for cyno T-cell binding of the parental monospecificanti-CD3. Column 4 shows the name of the aCD3:aBCMA bispecific antibody.Column 5 shows the picograms of IL-2 released by pan T-cells stimulatedby the bispecific antibody binding the BCMA protein coated on plastic atthe dose indicated. Column 6 shows the picograms of IL-6 released by panT-cells stimulated by the bispecific antibody binding the BCMA proteincoated on plastic at the dose indicated. Column 7 shows the picograms ofIL-10 released by pan T-cells stimulated by the bispecific antibodybinding the BCMA protein coated on plastic at the dose indicated. Column8 shows the picograms of IFN-γ released by pan T-cells stimulated by thebispecific antibody binding the BCMA protein coated on plastic at thedose indicated. Column 9 shows the picograms of TNFα released by panT-cells stimulated by the bispecific antibody binding the BCMA proteincoated on plastic at the dose indicated. Column 10 shows the EC₅₀ ofbispecific antibody-mediated U266 tumor cell lysis in presence of humanpan T-cells. Column 11 shows the percent lysis of U266 tumor cells inthe presence of bispecific antibody and human pan T-cells at a dose of333 ng/mL of bispecific antibody. Column 12 shows the protein bindingaffinity of the anti-CD3 arm of the bispecific antibody measured byOctet. Column 13 shows the MFI value for Jurkat cell binding of thebispecific antibody.

Example 5 Bispecific Antibody Characterization

Seven αCD3_Jam2:aBCMA bispecific antibodies, each with a unique anti-CD3arm and a common anti-BCMA arm, were tested for the ability to kill U266BCMA+ tumor cells through redirection of activated primary T cells. Inthis experiment U266 cells that express BCMA were mixed with activatedpan T-cells in a 10:1 E:T ratio along with the addition of bispecificantibody. Shown in FIG. 4 , the x-axis shows the concentration ofantibody used and the y-axis shows the % lysis of tumor cells 6 hoursafter addition of antibody.

A comparison of bispecific antibody-mediated tumor cell lysis activitywith IL-2 cytokine release is shown in the scatter plot of FIG. 5 . Thecorrelation between IL-2 production and U266 tumor cell lysis isR²=0.37. A comparison of bispecific antibody-mediated tumor cell lysisactivity with IFN-γ cytokine release is shown in the scatter plot ofFIG. 6 . The correlation between IFN-γ production and U266 tumor celllysis is R²=0.53. A comparison of bispecific antibody-mediated U266tumor cell lysis activity with anti-CD3 binding affinity is shown in thescatter plot of FIG. 7 . The correlation between U266 killing EC50 andprotein binding affinity is R²=0.93.

Example 6 Lysis of Tumor Cells

αCD3_F1 F:αBCMA bispecific antibodies were assayed for the ability tokill three different BCMA+ tumor cells and one BCMA-negative cell linethrough redirection of activated primary T cells. In this experiment,tumor cells were mixed with activated pan T-cells in a 10:1 E:T ratioalong with the addition of bispecific antibody. The results are shown inFIGS. 8A-6D. Panel A shows killing of RPMI-8226 cells, panel B showskilling of NCI-H929 cells, panel C shows killing of U-266 cells, andpanel D shows killing of K562 cells, a negative control. The x-axisshows the concentration of antibody used and the y-axis shows the %lysis of tumor cells 6 hours after addition of antibody.

The level of IL-2 cytokine release was measured after resting human Tcells were cultured with various tumor cell lines and increasing dosesof αCD3_F1F:αBCMA bispecific antibody. FIG. 9A shows IL-2 releasestimulated by RPMI-8226 cells, FIG. 9B shows IL-2 release stimulated byNCI-H929 cells, FIG. 9C shows IL-2 release stimulated by U-266 cells,and FIG. 9D shows IL-2 release stimulated by K562 cells, a negativecontrol.

The level of IFN-γ cytokine release was measured after resting human Tcells were cultured with various tumor cell lines and increasing dosesof αCD3_F1F:aBCMA bispecific antibody. FIG. 10A shows IFN-γ releasestimulated by RPMI-8226 cells, FIG. 10B shows IFN-γ release stimulatedby NCI-H929 cells, FIG. 10C shows IFN-γ release stimulated by U-266cells, and FIG. 10D shows IFN-γ release stimulated by K562 cells, anegative control.

The examples are put forth so as to provide those of ordinary skill inthe art with a complete disclosure and description of how to make anduse the present invention, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-38. (canceled)
 39. A method for treating a disease or condition,comprising administering to an individual in need an effective dose of amultispecific antibody comprising: (i) a first binding moiety that hasbinding specificity for human CD3δε, comprising: a heavy chain variabledomain comprising: A) a CDR1 sequence comprising GFTFDDYA (SEQ ID NO:29), a CDR2 sequence comprising ISWNSGSI (SEQ ID NO: 24), and a CDR3sequence comprising AKDSRGYGDYRLGGAY (SEQ ID NO: 41); or B) a CDR1sequence comprising GFTFHNYA (SEQ ID NO: 34), a CDR2 sequence comprisingISWNSGSI (SEQ ID NO: 24), and a CDR3 sequence comprisingAKDSRGYGDYSLGGAY (SEQ ID NO: 43); and a light chain variable domaincomprising a CDR1 sequence comprising QSVSSN (SEQ ID NO: 35), a CDR2sequence comprising GAS (SEQ ID NO: 38), and a CDR3 sequence comprisingQQYNNWPWT (SEQ ID NO: 45); and (ii) a second binding moiety that hasbinding specificity for a tumor-associated antigen (TAA) or atumor-specific antigen (TSA).
 40. The method of claim 39, wherein thedisease is an autoimmune disease.
 41. The method of claim 39, whereinthe disease is a cancer.